Novel oral multifunctional antioxidant prevents noise-induced hearing loss and hair cell loss.

Oxidative stress is a major contributor to noise-induced hearing loss, the most common cause of hearing loss among military personnel and young adults. HK-2 is a potent, orally-active, multifunctional, redox-modulating drug that has been shown to protect against a wide range of neurological disorders with no observed side effects. HK-2 protected cochlear HEI-OC1 cells against various forms of experimentally-induced oxidative stressors similar to those observed during and after intense noise exposure. The mechanisms by which HK-2 protects cells is twofold, first by its ability to reduce oxidative stress generated by free radicals, and second, by its ability to complex biologically active transition metals such as Fe+2, thus reducing their availability to participate in the Fenton reaction where highly toxic hydroxyl radicals are generated. For the rat in vivo studies, HK-2 provided significant protection against noise-induced hearing loss and hair cell loss. Noise-induced hearing loss was induced by an 8-16 kHz octave band noises presented for 8 h/d for 21 days at an intensity of 95 dB SPL. In the Prevention study, HK-2 was administered orally beginning 5 days before the start of the noise and ending 10 days after the noise. Treatment with HK-2 dose-dependently reduced the amount of noise-induced hearing impairment, reflected in the cochlear compound action potential, and noise-induced hair cell loss. In a subsequent Rescue experiment in which HK-2 was administered for 10 days starting after the noise was turned off, HK-2 also significantly reduced the amount of hearing impairment, but the effect size was substantially less than in the Prevention studies. HK-2 alone did not adversely affect HEI-OC1 cell viability, nor did it cause any adverse changes in rat body weight, behavior, cochlear function or hair cell integrity. Thus, HK-2 is a novel, safe, orally-deliverable and highly effective otoprotective compound with considerable potential for preventing hearing loss from noise and other hearing disorders linked to excessive oxidative stress.

Asteroid hyalosis: pathogenesis and prospects for prevention.

Asteroid hyalosis (AH) is a common degenerative process in which fatty calcium globules collect within the vitreous humour. The condition rarely causes visual disturbances, and surgical removal is only rarely required. The presence of AH has been associated with systemic diseases such as diabetes; however, research in this area has been hampered by the lack of an animal model of AH. Recently, we have reported that AH occurs in galactose-fed beagles that develop the advanced stages of diabetes-like retinopathy. Comparisons of vitreous humour containing asteroid bodies (ABs) collected from these galactose-fed beagles and vitreous samples from age-matched normal beagles without ABs indicate that ABs contain calcium and phosphorous. Subtraction analysis of chloroform extracts of the vitreous samples by electrospray mass spectroscopy resulted in the identification of the quasimolecular ion of 1,2-dipalmitoyl-glycero-3-phosphoethanolamine (DPPE) as the main component of ABs. Since several reports indicate that ABs are composed of lipid-calcium complexes, we have proposed that the main component of ABs in the galactose-fed dogs with AH is a quasimolecular ion of DPPE in which two molecules of DPPE are complexed through their phosphates groups with calcium. We suggest that these lipid components diffuse into the vitreous from the degenerating retinas of these dogs.

Method for isolating tight-binding inhibitors of rat lens aldose reductase.

Numerous animal studies indicate that aldose reductase inhibitors (ARIs) are beneficial for the prevention or amelioration of diabetic complications such as neuropathy, nephropathy and the ocular complications of cataract, retinopathy and keratopathy. To aid in the identification of novel potent ARIs, we have previously developed a screening method that is based on the formation of a non-covalent ternary tight-binding enzyme-inhibitor-nucleotide (AR-ARI-NADPH) complex that can be isolated using YM-10 filter units. Here, we report a modification of this method that permits us to rapidly identify tight binding ARIs that are isolated by denaturation from AR-ARI-NADPH complexes that are free of possible contamination resulting from the reaction of methanol with the YM-10 filter units. For the development of this procedure, nine structurally diverse ARIs were mixed with purified recombinant rat lens aldose reductase (RLAR) bound with NADPH to form tight-binding RLAR-ARI-NADPH complexes. These complexes were purified by high pressure Sephadex 75 size exclusion chromatography using ammonium acetate buffer and the formation of each complex was confirmed by electrospray ionisation mass spectrometry (ESI-MS). Each of the complexes was then denatured with methanol, rechromatographed on the size exclusion column, and the identity of the bound ARIs was confirmed by ESI-MS. The apparent ARI binding with aldose reductase to form a tight binding ARI complex appeared proportional to their IC50 values. This procedure allows for the rapid identification of tight binding ARIs with apparent IC50s<0.1 microm.

Selective pericyte degeneration in the retinal capillaries of galactose-fed dogs results from apoptosis linked to aldose reductase-catalyzed galactitol accumulation.

Galactose-fed dogs develop retinal capillary changes similar to diabetic retinopathy with pericyte degeneration as the initial lesion. This is followed by the formation of microaneurysms, hemorrhages, and some areas of acellularity. To investigate the mechanisms for selective pericyte degeneration, retinal capillary pericytes and endothelial cells isolated from beagle dog retina were cultured for 2 weeks in Dulbecco's modified Eagle's medium (DMEM) containing 50 mM D-galactose. Apoptosis was detected in pericytes but not endothelial cells by in situ terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labelling (TUNEL) staining and the DNA fragmentation assay on agarose gel electrophoresis. This apoptosis was prevented by the addition of the aldose reductase inhibitor AL 1576 to the culture medium containing galactose. Apoptosis was not observed when pericytes were similarly cultured in control DMEM medium. These data support the premise that the selective degeneration of retinal capillary pericytes observed in galactose-fed dogs is linked to increased aldose reductase activity in these cells.

Intrinsic inhibition of aldose reductase.

The development of aldose reductase inhibitors for the treatment of diabetic complications, such as cataract and retinopathy, has been of intense interest in the pharmaceutical community for the last 20 years. To date, aldose reductase inhibitors have been synthetically developed from leads obtained from in vitro screening studies. Recently, we have observed that mammalian tissues contain intrinsic inhibitors of aldose reductase, which may be used as potential drugs for treating diabetic complications with potentially less side effects than synthetic aldose reductase inhibitors. Intrinsic inhibitor(s) of aldose reductase have been observed in the methanolic extracts from rat and human kidneys and bovine lenses that were subjected to a number of chromatographic techniques, including counter current chromatography, flash chromatography, gel filtration and high pressure liquid chromatography. This inhibition results from a direct interaction between the inhibitor and enzyme. The intrinsic inhibitor, present in the lipophilic fraction of human kidney and bovine lens extracts, can easily penetrate into the lens to inhibit sugar alcohol formation. Intraperitoneal injection of partially purified bovine lens extract inhibited lens polyol formation in young rats fed 50% galactose diet.

Synthesis of potential aldose reductase inhibitors based on minimal pharmacophore requirements.

A series of 17 compounds were synthesized based on the premise that the minimal pharmacophore for aldose reductase inhibition requires the presence of both an aryl group and polar group connected by a linking structure. Three groups of compounds were synthesized, the first possessing an aniline-4-(2'-6'-methylbenzothiazole) or 2-aminobenzothiazole group as the aryl group, the second possessing a 2-naphthyl as the aryl group and the third possessing either a 4-(2-phenylthiazole) or 2-(5-2'-nitrophenylfuran) as the aryl group. In all three of these groups the carboxylate or its methyl ester are linked to the aryl group through various lengths of methylene carbons and amide or cinnamide groups. Optimal activity was observed when the carboxylic group was separated from the aryl group by a linking structure of five atoms in length. Both a double bond and an amide moiety are well tolerated in the linking structure.

Effect of bovine small intestine thioredoxin on aldose reductase activity.

Under oxidative stress mediated by H(2)O(2), significant activation of purified aldose reductase from bovine small intestine was observed in the presence of purified thioredoxin from bovine small intestine.

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis).

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 microM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123+/-10 microgram/10(6) cells (n=5) to 3.9+/-1.9 microgram/10(6) cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.

Determination of aldose reductase activity in the eye by localized magnetic resonance spectroscopy.

The polyol pathway plays an important role in the formation of diabetic complications of the eye. Due to variations in the pharmacokinetic properties of aldose reductase inhibitors and variations in the degradation of the blood-ocular barrier, it is often difficult to determine the proper intraocular levels of aldose reductase inhibitor required for inhibition of aldose reductase activity in ocular tissues. Utilizing localized magnetic resonance spectroscopy (MRS), the present method can determine adequate inhibition of aldose reductase activity in the lens by noninvasively measuring polyol pathway activity in the eye. New Zealand White rabbits, under anesthesia, were administered an intravitreal injection of 3-fluoro-3-deoxy-D-glucose (3FDG). Localized MRS was then used to assess polyol pathway activity by determining the levels of 3-fluoro-3-deoxy-D-sorbitol (3FS) and 3-fluoro-3-deoxy-D-fructose (3FF) metabolite formation from 3FDG in the eye. MRS was able to follow the loss of 3FDG from the vitreous into the anterior segment of the eye and particularly into the lens and aqueous. The primary metabolism of 3FDG observed by MRS was the formation of 3FS in the lens that is catalyzed by aldose reductase. Production of 3FS was linear in time and decreased with the oral administration of an aldose reductase inhibitor.

MRI of the human eye using magnetization transfer contrast enhancement.

PURPOSE: To determine the feasibility of using magnetization transfer contrast-enhanced magnetic resonance imaging (MRI) to track cataractous lens changes. METHODS: A fast spin-echo sequence was modified to include a magnetization transfer contrast (MTC) preparation pulse train. This consisted of twenty 8.5-msec sinc pulses, 1200 Hz upfield from the water resonance and 1.2-Hz power. The MTC preparation pulse was followed by acquisition through fast spin-echo imaging. The imaging parameters were number of excitations (NEX) = 1, echo time (TE) = 14 msec, recovery time (TR) = 2 sec, echo train length of eight echos, and a matrix size of 256 x 160. To reduce motion artifacts, the volunteers were asked to fixate on a blinking LED. Normal and MTC-enhanced images were acquired from normal volunteers and volunteers with nuclear or cortical cataracts. RESULTS: The eye was adequately imaged, with few motion artifacts appearing. The lens was well resolved, despite the short T(2). The cornea and ciliary body were also clearly visible. In the lens, resolution of the epithelium and cortex were enhanced with MTC. In addition, contrast-to-noise ratios were measured for each image. Examination of the contrast-to-noise ratio confirmed that MTC increased the contrast between the nucleus and cortex. Unenhanced MRIs showed significant differences between the cortex of normal volunteers and volunteers with cataracts. MTC-enhanced images improved the sensitivity to changes in the nucleus. CONCLUSIONS: In this preliminary study, we were able to use MTC-enhanced MRI to obtain high-contrast images of the human lens. Regular and enhanced MRIs detected statistically significant differences between normal and cataractous lenses.

Synthesis and aldose reductase inhibitory activities of novel N-nitromethylsulfonanilide derivatives.

A novel series of 14 N-nitromethylsulfonanilide derivatives were synthesized and evaluated for their ability to inhibit recombinant aldose reductase. Computational docking simulations provided a good explanation for the observed structure-activity relationships. Kinetic analysis of (2-fluoro-5-methyl-N-methyl)-N-nitromethylsulfonanilide, 11, one of the most potent compounds in this series with an IC50 = 0.35 M, showed uncompetitive inhibition. Subsequent in vitro culture studies of rat lenses with 11 indicated that this series of aldose reductase inhibitors are effective in either preventing or retarding sugar cataract formation associated with diabetes.

Vascular endothelial growth factor (VEGF) enhances the expression of receptors and activates mitogen-activated protein (MAP) kinase of dog retinal capillary endothelial cells.

Since the galactose-fed dog is an animal model that develops the advanced stage of proliferative retinopathy, the effects of vascular endothelial growth factor (VEGF) on cell growth, receptor expression and the activation of mitogen-activated protein (MAP) kinase pathway of dog retinal capillary endothelial cells were investigated. Dog retinal endothelial cells were cultured at 37 degrees C under 5% carbon dioxide atmosphere in CS-C medium supplemented with endothelial cell growth factor (ECGF). VEGF receptor expression was examined by RT-PCR, and activation of MAP kinase was examined with antibody against phospho-Elk-1 (Ser383). When growth factors were removed from the culture medium, cell survival of dog endothelial cells was significantly reduced. Addition of VEGF protected these cells from cell death induced by growth factor starvation. VEGF also enhanced tube formation in dog endothelial cells and increased the expression of two VEGF receptors, Flt-1 and KDR/Flk-1. Cells treated with VEGF also displayed the phosphorylation of the transcription factor, Elk-1. Addition of the tyrosine kinase inhibitor, genistein, eliminated VEGF-induced cell growth and Elk-1 phosphorylation. These data confirm that cell growth and tube formation of dog retinal capillary endothelial cells are stimulated by VEGF. VEGF also increases the expression of the receptors, KDR and Flt-1, and activates the p44/42 MAP kinase pathway.

Relative importance of aldose reductase versus nonenzymatic glycosylation on sugar cataract formation in diabetic rats.

The relative importance of sorbitol formation versus nonenzymatic glycosylation and advanced glycosylation end products (AGEs) on sugar cataract formation was examined in diabetic rats. Diabetes was experimentally induced in young, 50 g rats with streptozotocin, and aldose reductase inhibitors were administered in the diet for up to 8 weeks at concentrations of 0.06% for tolrestat or ponalrestat and 0.0125% for AL-1576. Cataract formation was monitored by hand-held slit lamp for up to 11 weeks. Lens polyol levels were monitored by GLC, glycosylated protein levels were spectrophotometrically determined, and AGE products were estimated by fluorescence measurements and ELISA. Sugar cataract formation was observed in all untreated diabetic rats while cataract formation was inhibited in all diabetic rats treated with the AR inhibitors. Lens sorbitol levels were reduced in all ARI-treated rats. Glycosylated lens protein levels were elevated in the diabetic rats, and these levels were not significantly lower in the non-cataractous lenses from ARI-treated diabetic rats. Fluorescence measurements of the lens proteins revealed increased lens AGE levels in all diabetic rats, and these were slightly reduced in the aldose reductase inhibitor treated diabetics. With ELISA, immunoreactive AGEs were only detected in cataractous lenses from the untreated diabetic rats. Immunoreactive AGEs were not detected in the clear lenses of the aldose reductase inhibitor treated diabetics or in the non-diabetic controls. These results support the concept that sugar cataract formation is initiated by the aldose reductase catalyzed intracellular accumulation of polyols and that these sugar cataracts can be prevented through inhibition of aldose reductase.

Isolation of a non-covalent aldose reductase-nucleotide-inhibitor complex.

A method for the isolation of an intact, non-covalent complex formed by the interaction of aldose reductase, NADP(H) nucleotide, and inhibitor has been developed to aid in the discovery and development of novel aldose reductase inhibitors. In the complexes isolated, both the carboxylic acid-containing inhibitor tolrestat and the spirohydantoin-containing inhibitor AL1576 (2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione) tightly bound in a 1:1 ratio to aldose reductase complexed with either NADPH or NADP+. Inhibitor binding to either the enzyme-NADP+ or enzyme-NADPH complex appeared to be equal and pH-dependent, with maximum binding observed at a pH range of 7 to 8.5 where the inhibitors are ionized. These results indicated that the charge state of the cofactor (NADPH vs NADP+) is not critical for inhibitor binding to aldose reductase. Molecular modeling studies suggested that His110 plays a crucial role in directing charged inhibitors containing either a carboxylate or an ionizable hydantoin group to the active site of aldose reductase by providing charge interaction.

Neutrophils in galactose-fed dogs: suppressed apoptosis and increased adhesion to retinal capillary endothelial cells.

Dogs fed a diet containing 30% galactose develop diabetes-like retinal capillary changes. As retinal capillary occlusion is commonly observed in diabetic retinopathy, neutrophil apoptosis and the interaction of neutrophils with retinal capillary endothelial cells were investigated. Neutrophils were isolated with Ficoll-Hypaque centrifugation from dogs fed a 30% galactose diet and dogs fed a normal, control diet containing 30% non-nutrient filler. Apoptosis of neutrophils was microscopically examined after incubation at 37 degrees C for 3 hours with either 100 U/mL tumor necrosis factor alpha (TNF-alpha), 2 microg/mL cycloheximide or 50 ng/mL phorbol 12-myristate 13-acetate (PMA). Neutrophil adhesion to dog retinal capillary endothelial cells was examined by counting the cells attached to the surface of endothelial cells after the incubation in the presence of either 100 U/mL TNF-alpha or 5 microg/mL lipopolysaccharides (LPS) at 37 degrees C for 3 hours. With all three stimulants TNF-alpha, cycloheximide and PMA, the rate of apoptosis was significantly lower for neutrophils isolated from galactose-fed dogs compared to control dogs fed a normal diet. Preincubation of neutrophils from control dogs in medium containing 30% galactose for 3 hours did not affect the rate of apoptosis. Neutrophil adhesion to retinal capillary endothelial cells induced by incubation in the presence of either 100 U/mL TNF-alpha or 5 microg/ml LPS was significantly higher with neutrophils isolated from galactose-fed dogs than those from control dogs. The data indicate that long-term galactose feeding is essential with development of various neutrophil dysfunctions. These neutrophil changes may contribute to the development of retinal microangiopathy associated with diabetes and galactosemia.

Aldose reductase, a key enzyme in the oxidative deamination of norepinephrine in rats.

The sympathoneural neurotransmitter norepinephrine (NE) is deaminated to 3,4-dihydroxymandelaldehyde (DHMAL) and subsequently converted to either 3,4-dihydroxymandelic acid (DHMA) or 3,4-dihydroxyphenylglycol (DHPG). In this study, we investigated the relative importance of aldose reductase versus aldehyde reductase in the formation of DHPG from DHMAL. The in vitro incubation of NE with aldose reductase in the presence of monoamine oxidase (MAO) resulted in the formation of DHPG, which was confirmed by mass spectrometry. Although aldehyde reductase also generated DHPG, its activity was much lower than that of aldose reductase. With northern blotting, the expression of both aldose reductase and aldehyde reductase was detected in rat superior cervical ganglia. However, with western blotting, only aldose reductase was immunologically detectable. Treatment of rats with aldose reductase inhibitors for 3 days increased the plasma level of DHMA. There was no correlation between the selectivity of inhibitors and effects on NE metabolite levels. A significant decrease in DHPG, however, was obtained only with an extremely high dose (9 mg/kg/day) of the nonselective inhibitor AL 1576. The present study confirmed that aldose reductase generates DHPG from NE in the presence of MAO. In rat sympathetic neurons, aldose reductase appears to be more important than aldehyde reductase for the formation of DHPG. However, when aldose reductase is inhibited, it appears that aldehyde reductase can compensate for the conversion of DHMAL to DHPG, indicating redundancy in the reduction pathway.